On the quest for the fluorescent “superheroes”

Published in Protocols & Methods
On the quest for the fluorescent “superheroes”

As a kid, I often argued with my friends about which superhero was the strongest and why. When I grew up, I replaced superheroes with FPs, and now I like to spend time on https://www.fpbase.org/ comparing different FPs (914 proteins to date) and helping my colleagues pick the right FP for various applications. Similarly to the superheroes, diverse existing FPs have a variety of “superpowers” that can be utilized by biologists and microscopists to reveal the inner mechanisms of life. For example, flavin- and bilin-binding FPs can be expressed under anaerobic conditions, allowing live cell imaging of obligate anaerobic bacteria.1,2 The large Stokes shift red FPs are characterized by the highest pH stability and are thus beneficial for imaging in an acidic environment occurring in lysosomes and mitochondria.3–5 Among all fluorescent “superheroes”, FPs derived from bacterial phytochrome proteins (BphPs) and cyanobacteriochromes (CBCRs) have a unique “superpower”, which is their red-shifted spectrum extending into the so-called “optical window”. Their superpower is the most useful for in vivo imaging of model organisms due to the reduced autofluorescence, light scattering and absorbance in the near-infrared (NIR) range of the visible spectrum. In addition, red-shifted light is less phototoxic and thus safer for long-term live cell imaging.

Unlike GFP-like FPs that form the chromophore autocatalytically, BphP- and CBCR-derived NIR FPs rely on exogenous chromophore biliverdin IXα (BV), one of the intermediates of heme metabolism6. Luckily, BV is present in, perhaps, most tissues and cell types in mammals. Alternatively, in other vertebrates, as well as invertebrates and bacteria, BV biosynthesis can be enabled by the expression of heme oxygenase, as heme is a ubiquitous biomolecule. However, due to the high variability of BV concentration in different cell types in combination with divergent affinities of BphP/CBCR-derived FPs to BV, intracellular brightness in one cell type is not readily correlated with that in another cell type.7 As a result, the molecular brightness of NIR FPs expressed in E. coli, typically used for selecting the brightest GFP-like FPs, cannot be reliably translated into intracellular brightness in mammalian cells. This discrepancy creates a need to test several FP candidates to pick those that perform optimally under actual experimental conditions. Since BphP/CBCR-derived FPs are often the proteins of choice for in vivo imaging, end users would be required to express FPs in model organisms. However, even a quick assessment of several FP candidates in vivo is significantly more laborious and time-consuming than careful characterization in cultured cells. On top of this, in the past decade, protein engineers have generated a large diversity of NIR FPs (>40 FPs), further complicating the end users’ task of defining a shortlist of FP candidates for testing. Systematic benchmarking of NIR FPs in various expression systems can aid end users in selecting the appropriate FPs for a given application, accelerating biological research. However, such a study can be a daunting task demanding lots of resources, effort, and time, while the desired outcome in terms of high-profile publication might not be guaranteed. Nevertheless, rapid technological progress in biosciences requires establishing standardized protocols and benchmarks that can serve as references to gauge the current state of the field and promote corresponding technologies.

To address this growing need, Nature Methods launched a new type of article, called Registered Report, which “describes a comprehensive comparison of the performance of established, related methods or tools in which the experimental design and data analysis plan is peer-reviewed and registered in a suitable repository prior to data collection” (see details here https://www.nature.com/nmeth/submission-guidelines/registered-reports). As one of the primary focuses of my lab is the development and application of fluorescent proteins, we immediately decided to use this unique opportunity to conduct a systematic and quantitative comparison of 22 most popular NIR FPs. When we proposed experimental design during Stage I, the referee feedback was crucial for refining a specific set of experiments and the corresponding data analysis approach. It also helped us plan all experiments carefully and thoughtfully. “The RR (Registered Report) process was well organized, and … rather flexible too, which is extremely helpful for authors” says Stavrini Papadaki, one of the authors who co-spearheaded the study. Knowing that the manuscript was accepted in principle took away some anxiety and allowed us to focus only on conducting experiments strictly according to the plan, thus promoting unbiased, fully transparent, and rigorous research. However, “the schedule itself was a bit overkill as the time needed to complete every experiment+analysis+figures was greatly underestimated, adding to the stress I felt” shares Stavrini. In retrospect, we understood that it was important to carefully estimate the timeline of the study by collecting feedback from every member of the team.

The ultimate goal of our Registered Report was to inform potential users about optimal NIR FPs for a variety of applications ranging from cell culture imaging to visualization of cellular and subcellular structures in vivo in model organisms, including mice, fish, and nematodes (see the corresponding article for the complete results and conclusions due to the limited space of the current format). I genuinely hope that the reported results will aid end users in selecting appropriate NIR FP saving their time, resource and streamlining experimental design. In addition, this study may lay a foundation for establishing validated benchmarks for NIR FPs that can be further used for gauging newly developed fluorescent proteins and dyes. Now, my lab, in close collaboration with many other research groups worldwide, is on the quest for new fluorescent “superheroes” with blue, cyan, green, yellow, and red capes.


  1. Chia, H. E., Zuo, T., Koropatkin, N. M., Marsh, E. N. G. & Biteen, J. S. Imaging living obligate anaerobic bacteria with bilin-binding fluorescent proteins. Curr. Res. Microb. Sci. 1, 1–6 (2020).
  2. Liang, G.-T. et al. Enhanced small green fluorescent proteins as a multisensing platform for biosensor development. Front. Bioeng. Biotechnol. 0, 1942 (2022).
  3. Erdogan, M., Fabritius, A., Basquin, J. & Griesbeck, O. Targeted In Situ Protein Diversification and Intra-organelle Validation in Mammalian Cells. Cell Chem. Biol. 27, 610-621.e5 (2020).
  4. Subach, O. M. et al. LSSmScarlet, dCyRFP2s, dCyOFP2s and CRISPRed2s, Genetically Encoded Red Fluorescent Proteins with a Large Stokes Shift. Int. J. Mol. Sci. 2021, Vol. 22, Page 12887 22, 12887 (2021).
  5. Vlaskina, A. M. ; V. ; et al. LSSmScarlet2 and LSSmScarlet3, Chemically Stable Genetically Encoded Red Fluorescent Proteins with a Large Stokes’ Shift. Int. J. Mol. Sci. 2022, Vol. 23, Page 11051 23, 11051 (2022).
  6. Piatkevich, K. D., Subach, F. V. & Verkhusha, V. V. Engineering of bacterial phytochromes for near-infrared imaging, sensing, and light-control in mammals. Chem. Soc. Rev. 42, 3441–52 (2013).
  7. Babakhanova, S. et al. Rapid directed molecular evolution of fluorescent proteins in mammalian cells. Protein Sci. 31, 728–751 (2022).

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